Feed formulations based on crop plants must typically be supplemented with specific amino acids to provide animals with essential nutrients which are necessary for their growth. This supplementation is necessary because, in general, crop plants contain low proportions of several amino acids which are essential for, and cannot be synthesized by, monogastric animals.
The seeds of crop plants contain different classes of seed proteins. The amino acid composition of these seeds reflects the composition of the prevalent classes of proteins. Amino acid limitations are usually due to amino acid deficiencies of these prevalent protein classes.
Among the amino acids necessary for animal nutrition, those that are of limited availability in crop plants include methionine, lysine, and threonine. Attempts to increase the levels of these amino acids by breeding, mutant selection, and/or changing the composition of the storage proteins accumulated in the seeds of crop plants, have met with limited success, or were accompanied by a loss in yield.
For example, although seeds of corn plants containing a mutant transcription factor, (opaque 2), or a mutant α-zein gene, (floury 2), exhibit elevated levels of total and bound lysine, there is an altered seed endosperm structure which is more susceptible to damage and pests. Significant yield losses are also typical.
An alternative means to enhance levels of free amino acids in a crop plant is the modification of amino acid biosynthesis in the plant. The introduction of a feedback-regulation-insensitive dihydrodipicolinic acid synthase (“DHDPS”) gene, which encodes an enzyme that catalyzes the first reaction unique to the lysine biosynthetic pathway, into plants has resulted in an increase in the levels of free lysine in the leaves and seeds of those plants. An increase in the levels of free lysine in the embryo results in reduced amount of oil in the seed. Further free lysine can be lost during the wet milling process reducing the feed value of the gluten product of the process.
The expression of the lysC gene, which encodes a mutant bacterial aspartate kinase that is desensitized to feedback inhibition by lysine and threonine, from a seed-specific promoter in tobacco plants, has resulted in an increase in methionine and threonine biosynthesis in the seeds of those plants. See Karchi, et al.; The Plant J.; Vol. 3; p. 721; (1993). However, expression of the lysC gene results in only a 6–7% increase in the level of total threonine or methionine in the seed. The expression of the lysC gene in seeds has a minimal impact on the nutritional value of those seeds and, thus, supplementation of feed containing lysC transgenic seeds with amino acids, such as methionine and threonine, is still required.
There are additional molecular genetic strategies available for enhancing the amino acid quality of plant proteins. Each involves molecular manipulation of plant genes and the generation of transgenic plants.
Protein sequence modification involves the identification of a gene encoding a major protein, preferably a storage protein, as the target for modification to contain more codons of essential amino acids. An important aspect of this approach is to be able to select a region of the protein that can be modified without affecting the overall structure, stability, function, and other cellular and nutritional properties of the protein.
The development of DNA synthesis technology allows the design and synthesis of a gene encoding a new protein with desirable essential amino acid compositions. For example, researchers have synthesized a 292-base pair DNA sequence encoding a polypeptide composed of 80% essential amino acids and used it with the nopaline synthetase (NOS) promoter to construct a chimeric gene. Expression of this gene in the tuber of transgenic potato has resulted in an accumulation of this protein at a level of 0.02% to 0.35% of the total plant protein. This low level accumulation is possibly due to the weak NOS promoter and/or the instability of the new protein.
Tobacco has been used as a test plant to demonstrate the feasibility of this approach by transferring a chimeric gene containing the bean phaseolin promoter and the cDNA of a sulfur-rich protein Brazil Nut Protein (“BNP”), (18 mol % methionine and 8 mol % cysteine) into tobacco. Amino acid analysis indicates that the methionine content in the transgenic seeds is enhanced by 30% over that of the untransformed seeds. This same chimeric gene has also been transferred into a commercial crop, canola, and similar levels of enhancement were achieved.
However, an adverse effect is that lysine content decreases. Additionally, BNP has been identified as a major food allergen. Thus it is neither practical nor desirable to use BNP to enhance the nutritional value of crop plants.
Thus, there is a need to improve the nutritional value of plant seeds. The genetic modification should not be accompanied by detrimental side effects such as allergenicity, anti-nutritional quality or poor yield.